Project Summary The objective of this proposal is to determine the contribution of active DNA demethylation pathways to epigenetic reprogramming during mammalian germline development. DNA methylation in the form of 5- methylcyosine (5mC) serves as an essential epigenetic regulator of gene expression and cellular identity. Dysregulation of genome 5mC levels contributes to a number of human developmental disorders, including Rett syndrome, juvenile cancers, and imprinting disorders such as Beckwith-Wiedemann syndrome. While DNA methylation pathways are well defined, the mechanisms controlling 5mC removal are poorly understood. Members of the Ten-eleven Translocation (TET) family of enzymes regulate active DNA demethylation through the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). These oxidized residues are not recognized by maintenance methyltransferases, resulting in their loss over several rounds of cellular division in a process known as ?active modification with passive dilution? (AM-PD). Alternatively, 5fC and 5caC may be targeted for cleavage by thymine DNA glycosylase (TDG), triggering base excision repair to restore the unmodified cytosine. This process is referred to as ?active modification with active removal? (AM-AR). Although previous work suggests the AM-PD and AM-AR pathways carry-out distinct functions, researchers have lacked the necessary tools to distinguish between the two pathways in vivo. To that end, our collaborators have developed novel TET mutants proficient for the generation of 5hmC but not 5fC or 5caC, the necessary substrates for AM-AR demethylation. Using an orthologous knock-in model for mouse Tet1, Aim 1 will test how AM-AR deficiency affects germline development. Genome-wide 5mC and 5hmC levels will be measured in TET1 mutant germ cells an gametes and correlated with transcriptomic RNA levels determined by RNA-seq. To test the hypothesis that loss of the AM-AR pathway leads to female subfertility, histological assays will also be used to track oocyte maturation in TET1 mutant mice. Aim 2 will use a well-characterized iPSC model system that is dependent upon the TET family of enzymes to determine functional differences between the AM-AR and AM-PD pathways. To test whether the AM-PD pathway is sufficient to drive iPSC reprogramming, TET triple-knockout mouse embryonic fibroblasts will be transduced with AM-AR-deficient Tet1 and subjected to OSK reprogramming. Additionally, based on our hypothesis that AM-AR demethylation promotes broad epigenetic changes through the recruitment of histone modifiers and chromatin remodelers, chromatin immunoprecipitation experiments will be performed to monitor for changes in regulatory histone marks at genes important for iPSC reprogramming. Together, these Aims will elucidate the distinct roles of DNA demethylation pathways in regulating cellular identity and mammalian development, and may point to novel functions for the AM-AR pathway in promoting epigenetic reprogramming beyond 5mC erasure.